Native Pyrococcus furiosus ADP-dependent Hexokinase

Hexokinase, a key intracellular enzyme, is required for glucose metabolism. Hexokinase I, II, III, and IV are the four major isozymes of the hexokinase enzyme family. Type IV is also known as glucokinase. Their distribution, functions, and glucose affinity differ. Hexokinase I is predominantly expressed in brain tissue, where it is extremely affinistic for glucose and has developed to provide the tissue's energy requirements on a constant basis. Hexokinase II is a glycolysis regulator that is widely distributed in adipose and muscle tissues. It is involved in the direct delivery of ATP and is firmly attached to the outer mitochondrial membrane. Hexokinase III, although widely expressed in vivo, its specific physiological function is not well understood. Hexokinase IV (Glucokinase): expressed mainly in liver and pancreatic β-cells, it has a low affinity for glucose but its high Km value makes it suitable to act under high glucose It acts under hyperglycemic conditions to regulate insulin secretion and hepatic glycogen synthesis. Hexokinase usually consists of two major structural domains, each capable of binding ATP and glucose molecules. In enzymatic reactions, Hexokinase binds to glucose through its N-terminus and subsequently catalyzes a phosphate group transfer reaction through its C-terminus. This process converts the glucose molecule to glucose-6-phosphate, which prevents it from returning through the cell membrane, thus locking the glucose inside the cell for further metabolism. In addition to its catalytic function, Hexokinase also plays a crucial role in the regulation of cellular metabolism. when Hexokinase binds to the outer mitochondrial membrane, it can directly utilize ATP produced by the mitochondria, thus accelerating the phosphorylation reaction. The binding and release of Hexokinase is also regulated by different metabolite concentrations, especially when the intracellular ADP and AMP levels rise, Hexokinase activity increases significantly and promotes glucose metabolism to generate more energy. Hexokinase activity is regulated by a variety of factors including substrate concentration, enzyme inhibitors, and intracellular energy status. Glucose and ATP are two key substrates for Hexokinase, and their concentrations directly affect enzyme activity. In the low glucose state, the activity of Hexokinase is reduced to prevent unnecessary energy waste, while in the high glucose state, the activity of Hexokinase is increased to promote rapid sugar metabolism. Meanwhile, Hexokinase is feedback inhibited by its reaction product glucose-6-phosphate. When intracellular glucose-6-phosphate accumulates, it binds to Hexokinase and prevents further binding of glucose to the enzyme, thereby regulating metabolic flow. In addition, the intracellular ATP/ADP ratio is an important signal for Hexokinase regulation. When intracellular energy levels are low, AMP levels rise and Hexokinase activity increases, promoting glycolysis to produce more ATP. conversely, when ATP levels are high, Hexokinase activity decreases, reducing glucose consumption.

In plants, Hexokinase is not only involved in sugar metabolism, but also plays an important role in photosynthesis, energy perception, and development. Plant cells use photosynthesis to convert light energy into chemical energy to produce glucose, and Hexokinase catalyzes the phosphorylation of glucose to participate in glycolysis and provide energy to plant cells. More importantly, Hexokinase also acts as a sensor for plants to sense the glucose level in the cell and regulates the expression of a series of genes related to plant growth and development. For example, when the sugar level changes in the external environment, Hexokinase is able to sense such changes and influence the processes of root development, seed germination and leaf senescence in plants by regulating gene expression. In addition, Hexokinase also plays an important role in plant stress tolerance, helping plants to cope with unfavorable environmental conditions such as drought and salt stress. In human cells, the role of Hexokinase is even more extensive and complex. It not only participates in sugar metabolism and provides a direct energy source for cells, but also plays an important regulatory role in various physiological processes. First, Hexokinase maintains intracellular ATP supply by regulating the balance between glycolysis and oxidative phosphorylation. In high-energy-demanding tissues such as the brain, heart, and muscles, Hexokinase ensures efficient utilization of glucose in these organs, thus supporting their normal functional operation. Secondly, Hexokinase is associated with processes such as apoptosis and oxidative stress response. Particularly in cancer cells, due to the overexpression of Hexokinase II, cancer cells can obtain large amounts of energy through rapid glycolysis to support their abnormal proliferation. Therefore, Hexokinase is also a target for many metabolic diseases and cancer therapeutic studies.

Figure 1. Role of HXK in Plant Physiology (Source: Granot D, et al., 2014)

The role of Hexokinase in metabolic diseases is of great interest, especially in pathological conditions such as diabetes and cancer. In patients with type 2 diabetes, Hexokinase IV activity is significantly affected. The function of this enzyme in the pancreas is to sense blood glucose levels and regulate insulin secretion. When glucose enters the pancreatic beta cells, Glucokinase is responsible for catalyzing its phosphorylation, prompting insulin release. However, in diabetic patients, due to abnormal function or reduced activity of this enzyme, blood glucose sensing is diminished, leading to insufficient insulin secretion, which exacerbates the hyperglycemic state. In addition to this, many tumor cells exhibit abnormally high Hexokinase activity, especially Hexokinase II. Its overexpression is closely related to the Warburg effect (tumor cells preferentially generate energy through glycolysis, even under aerobic conditions). Hexokinase II significantly enhances the rate of glucose metabolism by binding to mitochondria, providing the rapid proliferation of tumor cells by providing sufficient energy and metabolic intermediates. Therefore, Hexokinase II is regarded as a potential therapeutic target for anticancer treatment, and by inhibiting its activity, it can effectively reduce the energy supply of tumor cells and inhibit their growth. Due to its important role in multiple metabolic pathways, Hexokinase is considered a potential therapeutic target for a variety of metabolism-related diseases. Currently, researchers are developing specific inhibitors or activators against Hexokinase for the treatment of diabetes, cancer and other diseases. For example, in cancer therapy, Hexokinase inhibitors can inhibit tumor growth and spread by blocking energy metabolic pathways in tumor cells. Meanwhile, modulation of Hexokinase activity may also be used for the prevention and treatment of metabolic syndrome, cardiovascular diseases, etc. Overall, Hexokinase not only plays a key role in basic metabolism, but its importance in diseases has also made it a focus of research in multiple fields. In the future, with the in-depth study of its structure and function, the application of Hexokinase in clinical treatment will be more promising.

Figure 2. HK2 Regulation Pathways in Tumor Cells (Source: Ciscato F, et al., 2021)

Pyrococcus furiosus hexokinase
ADP-dependent hexokinase
P. furiosus hexokinase
PfHK (Pyrococcus furiosus Hexokinase)